Preparing Lambda DNA

1- Coliphage lambda DNA is a widely used vector for recombinant DNA. The middle
third of its 48,000 bp contains no genes required for lytic growth and is, therefore,
replaceable. The usual recombinant lambda DNA contains 80% lambda vector DNA
and 20% insert, as compared to a usual cosmid DNA that contains 10% vector and
90% insert.

Wild-type lambda is not very lytic, compared to coliphages T4 or T7. Recombinant
lambda phage are usually less lytic than wild-type. We recommend growing recombinant lambda phage on large (150-mm diameter) plates, rather than in liquid
culture. Beginners, in particular, may not be aware sufficiently that in liquid culture
E. coli can easily overgrow phage lambda, rather than the desired
vice versa.

2- Traditionally, the E. coli host for lambda is grown on NZY medium. This medium
is not as rich as LB (and many other) medium, and
E. coli growth is slower, making it even less likely that
E. coli will overgrow the phage.

The lambda receptor on the bacterial cell outer surface is part of the maltose-usage pathway. To induce the receptor to high levels (102-103 receptors per cell), maltose is added to a final concentration of 0.2% in the medium.

When growing lambda on plates to prepare DNA (as opposed to picking plaques or
titering), agarose is substituted for agar. Agarose is much more expensive than
agar, but does not contain impurities that inhibit enzymes. Lambda DNA prepared
from agar plates may not be digestible by restriction enzymes because of the
presence of inhibitors that have leeched from the agar.

3- The host for lambda is E. coli C600 and its subsequent variants. To avoid complications, the E. coli C600 strain is usually restriction-negative:
r-m- or r-m+.

For our purposes, we find it useful to start with a fresh
E. coli colony (on NZY or LB plates). The colony is picked into 5 ml of NZY medium plus 0.2% maltose and
placed on a wheel at 37°C for approximately 4 hr. (This time will be much longer if
the colony is from an old plate or has been stored at 4°C or frozen.) Using sterile
conditions, the culture is transferred to a sterile 15-ml conical tube. The
E. coli are pelleted by centrifugation in an SS34 rotor with adaptors in an RC-5B centrifuge
(or equivalent) for 8 min at 4 K rpm (or equivalent). The medium is poured off
gently. The bacterial pellet is resuspended in 4-6 ml of 0.01
M MgSO4 by pipetting up and down (using sterile conditions), NOT by vortexing. The concentration of
bacteria is adjusted to yield an "optical density" (really, light scattering) of 2.0 at
wavelength 600 nm. E. coli prepared in this way can be stored at room tempera
ture and used for up to one week. To resuspend the
E. coli, gently pipet the cells. DO NOT VORTEX. Cells prepared in this manner are employed at values of 10 µl
per small or 20 µl per large plate.

4- At times, we shall refer to the procedures published in Sambrook, Fritsch, and
Maniatis, in "Molecular Cloning: a laboratory manual", pp. 2.60-2.79, second edi
tion, 1991, as the "CSH" procedure or the "CSH" book.

One common source of confusion when comparing methods for growing lambda
concerns "SM buffer" and whether or not it contains gelatin. In our protocol, "SM
buffer" refers solely to Tris buffer with magnesium salt and NaCl. If gelatin is to be
added, we state so explicitly in the few cases where we use it.

5- The start of any procedure for preparing lambda DNA is to pick a single, well
-isolated plaque. (A lambda plaque contains phage and lysed bacteria and appears
relatively "clear" against the bacterial lawn.) For the Olson frozen lambda stocks,
we use sterile technique to scrape a small amount (approximately a toothpick-full)
of frozen stock into 1 ml of SM buffer plus gelatin. (The gelatin helps stabilize the
phage.) From this one ml, we make 10-fold dilutions (in SM buffer plus gelatin)
through 10-4. In general, we plate 1, 10, and 100 µl of the 10
-3 and 10-4 dilutions.

There is significant preparation time. NZY agar plates should be placed at 37°C, so
that they are warm when used. NZY top agar (3 ml per plate) should be melted
and placed at 50°C (just warm enough to keep it liquid, and not so hot as to kill
the E. coli). E. coli (10 µl) and 0.2 ml SM buffer (no gelatin) should be mixed in a
small tube, one tube per agar plate.

Phage are added to the E. coli and SM buffer, and the tubes are placed on the
37°C wheel for 20-30 min. The kinetics of lambda attachment to its receptor are
fairly slow; the 20 min incubation allows time for this reaction to occur. Add the 3
ml of top agar to the mixture of phage and bacteria. Vortex for 10 sec. Pour onto
an agar plate. Rock the plate by hand to reach an even distribution of top agar.
Allow the top agar to solidify at room temperature (approximately 10 min). Place
the inverted plates at 37°C overnight. The next day, at least one plate should
have well-isolated plaques. If there are too many plaques, start over with more
dilution. If there are too few plaques, start over with less dilution.

For picking plaques, we find the following procedure simple and straightforward.
Autoclave inverted Pasteur pipets (without cotton plugs). Holding the narrow end,
punch out a plaque with the (relatively) wide-mouth end. The agar stays in the
pipet and can be expelled into a sterile eppendorf tube by a wrist flick. Add 0.5 ml
of SM buffer (no gelatin) and 2 drops of chloroform (the chloroform stops the
E. coli from growing). To elute the phage, allow the eppendorf tube to stand for 2-4
hr at room temperature or at 4°C overnight.

6- A phage stock is made from the isolated plaque. Again, NZY agar plates are at
37°C; NZY top agar is at 50°C; 0.2 ml of SM buffer plus 10µl of
E. coli are in small tubes. Add 5-20 µl (or more) of phage, depending on plaque size, to the
E. coli and SM buffer. Place on a 37°C wheel for 20-30 min. Add NZY top agar and vor
tex for 10 sec. Pour onto a warm NZY agar plate. After the top agar hardens
(approximately 10 min), invert the plate and place at 37°C overnight.

The next morning the plates should show a fully infected, disrupted
E. coli lawn. If you see individual plaques, the inoculum was too low. Toss the plates, and repeat
the infection with a larger volume of the plaque. If the infection looks confluent,
add 5 ml of sterile SM buffer (no gelatin). Make sure that the plate is completely
covered with buffer. Gently rotate or rock the plates at room temperature for 1-2
hr. Harvest the buffer (using a sterile pipet) into a sterile conical 15-ml tube. The
"milky" appearance is caused by the presence of bacterial debris. Add 0.2 ml of
chloroform and vortex for 10 sec. Spin the tube in an SS34 rotor with adaptors,
RC-5B centrifuge (or equivalent), at 4 K rpm for 8 min. Decant the clear yellow
supernatant into a fresh, sterile, 15-ml conical tube. Add a drop of chloroform
(the chloroform is to stop the growth of residual
E. coli) and store at 4°C. These stocks are stable for several months at 4°C. Frozen (-70°C) stocks can be made
by mixing 0.1 ml DMSO with 1.4 ml of lambda phage stock.

7- The phage stocks of Olson lambda are usually in the range 10
5-106 pfu (plaque-forming units) per µl. These numbers are lower than those given in CSH, pg. 2.65;
the Olson lambda phage grow very poorly. Our stocks have been as low as 2x10
3 and as high as 8x107 pfu per µl. The general correlation is that phage that pro
duce small plaques tend to yield stocks below 105 pfu per µl; phage that produce
(relatively) large plaques tend to yield stocks over 10
6 pfu per µl; but the correlation is not absolute.

The lambda stocks are diluted ten-fold (as appropriate: 10
-2, 10-3, etc.) in SM buffer plus gelatin. NZY agar plates are at 37°C. NZY top agar is melted,
aliquoted, and maintained at 50°C. 0.2 ml of SM buffer plus 10 µl of
E. coli are in small tubes. Appropriate amounts (e.g., 1, 10, 100 µl) of each appropriate dilution
are added to the bacteria. The tubes are placed on the 37°C wheel for 20-30 min.
3 ml of top agar are added to the phage and bacteria; the tube is vortexed for 10
sec, and poured onto a warm agar plate. Distribute the top agar evenly on the
plate while rocking (rotating, swirling) by hand. Allow the top agar to harden at
room temperature (approximately 10 min). Invert the plates and place at 37°C
overnight.

The next morning look at the the plaques. They are usually of a reasonable size to
count. However, if the plaques are very small (which does occur with the Olson
lambdas), leave the plates at 37°C and check the size of the plaques hourly.
Count the plaques on plates where the number is between 50 and 300. A mini
mum of 100 is needed for reasonable statistical reliability; a plate with over 200
plaques may yield an incorrectly low number, as plaques overlap. How much
accurracy you need is up to you. Calculate the titer of the stocks.

8- With the preparation of titered phage stocks derived from a single plaque, all
the reagents are in hard to prepare lambda DNA. In addition to titered phage
stocks, you need large (150-mm) NZY agarose plates, NZY top agarose, and
E. coli C600 in 0.01 M magnesium sulfate. We switch from agar to (the much more ex
pensive) agarose to avoid the umwanted impurities present in agar. We usually
use two 150-mm plates for growth of each original phage plaque. The large NZY
agarose plates are equilibrated at 37°C. NZY top agarose is at 50°C. Place 0.2 ml
of SM buffer in a small tube and add 20 µl of
E. coli (double the amount for a small plate). For poor-growing lambda, such as the Olson recombinants, we add 50,000
pfu to the tube. However, this number can vary greatly: higher for very poor
-growing phage and much lower for phage which grow well. You may have to test
several phage inputs. The goal is to achieve confluent lysis, maximum phage pro
duction, without blowing away the E. coli prematurely. Add the lambda to the
bacteria and SM buffer and place the tube on the 37°C wheel for 20-30 min. Add
5 ml (an increased amount for the large plates) of top agarose; vortex for 10 sec,
and pour onto the warm 150-mm plate. Rotate (rock, swirl) the plate by hand to
achieve an even distribution of top agarose. Allow the top agarose to harden
(approximately 10 min) and invert the plate at 37°C overnight.

The next morning the plates should show confluent lysis. If individual plaques are
visible, you may not achieve a usable amount of phage DNA. Start over at a higher
input. We find that two confluently-lysed large plates will yield adequate amounts
of DNA, even for poorly-growing lambdas.

9- Add 8 ml of SM buffer to each plate. Make sure the buffer covers the entire
plate. Sterile conditions are no longer necessary, but neatness always counts.
Rotate gently (or rock gently) at room temperature for 1 to 2 hr. The phage will
elute into the buffer.

Decant (pipette) the buffer into a 15-ml conical tube (or equivalent): 5-6 ml/plate.
Two large plates of the same phage are combined, as appropriate. Centrifuge the
tube in an SS34 rotor with adaptors, RC-5B centrifuge (or equivalent) for 8 min at
4 K rpm (or equivalent). The debris will pellet, and most of the phage will reamain
in the supernatant. Decant the supernatant (9-12 ml) to an Oak Ridge tube.

10- In this next , crucial step, DNase I is added to digest contaminating
E. coli DNA. Lambda DNA, inside the phage particle, is protected from digestion. The
integrity of the phage requires the presence of magnesium ions, and DNase activity
requires magnesium ions, which are provided in SM buffer. Add 0.1 ml of DNase I
(100 µg per ml; RNase-free) and mix gently. Place the tube at 37°C for 1-2 hr.
(No attempt is made to remove RNA, and, in fact, RNA is used as a carrier for the
DNA.)

11- Inactivate DNase I activity and disrupt the phage particles by the additions of
0.5 ml of 0.5 M EDTA, pH 8, and 0.5 ml of 10% SDS. Mix gently. (The removal of
magnesium ions by the EDTA also causes the polysomes and ribosomes to fall
apart. This helps during phage DNA purification.)

13- Concentrate the DNA, and remove traces of phenol and chloroform, by alcohol
precipitation. Add two-volumes of cold (95-100%) ethanol. Vortex for 10 sec to
mix. Centrifuge in an SS34 rotor, RC-5B centrifuge (or equivalent), for at least 30
min at 12 K rpm at 10°C. Gently decant the supernatant. (We pour the alcohol
off carefully while watching that the precipitate does not move.) To remove ex
cess salt, gently add 10 ml of cold 70% ethanol. Do not disturb the nucleic acid
precipitate. Centrifuge for 10 min (or more) at 12 K rpm at 10°C. Gently decant
the supernatant. Add 10 ml of cold (95-100%) ethanol. Centrifuge for 10 min
utes at 12 K rpm at 10°C. Gently decant the supernatant. Drain briefly and air
dry briefly. Our experience with the Olson lambda DNAs is that the amount of
precipitate is highly variable. Most of the precipitate is RNA.

14- Dissolve the precipitate in 2.5 ml of TE. This process may take longer than 1
hr at room temperature. Add 0.1 ml RNase (1 mg/ml, DNase free) and mix gently.
(At this step, the contaminating RNA is removed by digestion). Place at 37°C for
1-2 hr. Add 0.1 ml of proteinase K (1 mg/ml, nuclease-free) and incubate at 37°C
for 1-2 hr. Contaminating protein, including RNase and proteinase K itself, is re
moved by digestion. Cool to room temperature.

16- The Centricon takes the place of alcohol precipitation and/or dialysis to
change buffer and concentrate the DNA. The Centricon works annoyingly slowly,
but the recoveries are excellent. Read the manufacturer's (Amicon Co.) instruc
tions. The plastic that composes the Centricon is resistant to traces of phenol and
chloroform but is easily damaged by isoamyl alcohol. Many recipies and commer
cially available phenol:chloroform add isoamyl alcohol as an anti-foam reagent. Do
not use those recipes/products, or do not use the Centricon.

We use a Centricon-30 spun at 5 K rpm in an SS34, RC-5B centrifuge at 10°C.
Other rotor/centrifuge combinations may have a different maximum speed; check
Amicon's specifications. The choice of 10°C, rather than 4°C, is to avoid icing-up.

Under these conditions, a 2.5 ml sample will pass through the filter in about 30-60
min. We load the sample completely, do three 2.5 ml TE washes, and then spin for
an additional 2 hr to reach minimum volume (usually 50-60 µl).

17- For the Olson lambda DNAs, we use 7 µl of the DNA to double-digest and run
on a 1% agarose gel in 0.5xTBE. If all has gone well, the restriction enzyme cleav
age pattern matches Olson's.

0.5 M EDTA, pH 8.0: EDTA is sold as the disodium salt. In solution, Na
2EDTA has a pH near 5 and a saturation limit around 0.2
M. To achieve 0.5 M, NaOH needs to be added in small aliquots.

10% SDS: 10 g of sodium dodecyl sulfate dissolved in water to a final volume of
100 ml.

Ethyl alcohol: 95 or 100% and 70%, both at -20°C.

Phenol: Standard commercial phenol is quite dirty by molecular biology standards
and must not be used. Several companies sell re-distilled phenol of "nucleic acid"
or "molecular biology" grade. We are using Amresco's saturated phenol, which is a
liquid, and comes with a small bottle of TE buffer to be added to the phenol. We
want buffer-saturated phenol. Add the buffer to the phenol and shake. Allow to
stand overnight in a refrigerator. Use an amber bottle to avoid light. We want to
avoid oxidation of the phenol to produce quinones, which are highly reactive and
detrimental to DNA. A reminder: a saturated solution cannot be created in an
hour. At saturation, the upper phase is excess buffer.

Chloroform (CHCl3): any brand at A.C.S. purity is fine.

Phenol: Chloroform (1:1): Mix one volume of buffer-saturated phenol with the
same volume of CHCl3 in an amber bottle. Shake and place in the refrigerator
overnight. The upper phase is excess buffer. We make our own mixture, because
Amresco's phenol: chloroform contains isoamyl alcohol as an anti-foam reagent,
and isoamyl alcohol dissolves the plastic Centricon.

For this procedure, the following reagents are required:

0.01 M MgSO4: used to resuspend
E. coli C600. We use A.C.S. grade MgSO4·7H
2O. Sterilize by autoclaving or filtration.

DNase I (free of RNase): Several companies sell this reagent. Our stock solu
tion is 100 µg/ml in 0.01 M Tris, pH 8.0. We purchase DNAse I from Boehringer
-Mannheim. The stock is aliquoted, frozen (-20°C), and thawed as needed. (no
EDTA) Careful: DNAse I is very heat labile and is easily inactivated at room tem
perature.

RNAse (free of DNAse): We purchase this enzyme from Boehringer-Mannheim.
(Even the smallest amount of contaminating DNAse will degrade your precious
DNA.) The RNAse is aliquoted and frozen (-20°C).

Sterilize by autoclaving. For agar plates (100-mm plates used for counting
or isolating plaques), add 15 g of agar per liter before autoclaving. For top agar,
add 7 g of agar per liter before autoclaving. For ease of handling, we prepare top
agar in 100-200 ml batches, rather than 1 l batches. For large (150-mm) agarose
plates, used when growing lambda to prepare DNA, add 15 g of agarose per liter
before autoclaving; top agarose requires 7 g of agarose per liter before autoclav
ing. Unless you have an unlimited supply budget, you should use agarose ony
where essential. We make both NZY-agar and NZY-agarose plates and top media to
save money.

Maltose (20%): Dissolve 20 g of maltose in a final volume of 100 ml. Sterilize
by filtration. Maltose, as all sugars, tends to become caramel upon autoclaving.

SM buffer: We do not consider gelatin to be a component of SM buffer. In this
usage, we differ from the CSH book. Throughout our text, SM buffer is composed
of: per liter:

NaCl 5.8 g

MgSO4·7H2O 2 g

1 M Tris, pH 7.5, 50 ml

(The CSH book uses the nomenclature "Tris·HCl", which is not technically correct.
We start with Tris·OH and add HCl to pH 7.5. Therefore, our Tris buffers do not
have additional sodium ions. Incidentally, Tris·OH is half of the cost of Tris·HCl.)

Gelatin (2%): We purchase a sterile 2% gelatin solution from Sigma (cat. # G
-1393) or other supplier. It's inexpensive, and we use very little. You could save
money by making this solution yourself. 2% gelatin is not quite a liquid at room
temperature. We warm the bottle gently in a microwave oven just before use.
Gelatin is added to SM buffer (5 ml/liter)when the combined buffer is used to
stabilize phage. Throughout our text, when gelatin is to be added, we state so
explicitly.